CN105572351B - Based on Electrochemiluminescince fungal detection micro-fluidic chip - Google Patents
Based on Electrochemiluminescince fungal detection micro-fluidic chip Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 128
- 230000002538 fungal effect Effects 0.000 title claims abstract description 11
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
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Abstract
The invention provides a kind of based on Electrochemiluminescince fungal detection micro-fluidic chip, detection substrate including carrying detection unit, the detection unit includes connected sample inlet area, memory block, detection zone and the waste of sequence, the detection unit rounded array on the detection substrate arranges and shares the sample inlet area in array center, and the detection zone is loaded with microelectrode.Flexibly convenient, sensitivity and flexibility are high for the invention operation.
Description
Technical field
The invention belongs to micro-fluidic chip design field, and in particular to a kind of immune inspection for fungal detection
Micrometer fluidic chip.
Background technology
In recent years, it is widely used with a large amount of abuses and immunosuppressor of antibiotic in clinical, aggressive deep is true
The incidence and case fatality rate of bacterium infection are higher and higher.Wherein, aspergillus, cryptococcus and candida albicans have been increasingly becoming clinically several
The important pathomycete of kind.Invasive bacterial infection detection at present has following drawback：Experimental period is longer, verification and measurement ratio is relatively low,
It consumes sample and amount of reagent is big, detection device is complicated, of high cost, the professional testing staff of needs is operated.Aggressive bacterium sense
Feelings of catching an illness make much progress, and traditional detection technique often causes to fail to pinpoint a disease in diagnosis, mistaken diagnosis, causes to affect the state of an illness adversely.Micro-fluidic chip, due to
The reagent and the volume very little (about 10 of sample manipulated-9~10-18L), improve detect reaction rate while, allow it is artificial because
Influence of the element to detection process be reduced to it is minimum, can be fabulous the drawbacks of evading traditional detection.Detection antigen is current clinical public
The effective ways of quick early diagnosis bacterium infection recognized.Aspergillus, cryptococcus and the Exoantigen of candida albicans are respectively
Galactomannans antigen, capsular polysaccharide antigen and mannan antigen can be found in infection early stage from blood samples of patients.
It is a kind of widest method of purposes in a kind of current biological detection method based on immune response detection antigen,
With high sensitivity and specificity.It is broadly divided into enzyme linked immunosorbent assay, immunofluorescence assay, electrochemical process, chemistry
The detection method that shines and electrochemical luminescence detection method.Wherein electrochemical luminescence detection method, due to its high sensitivity, wide dynamic
The features such as concentration-response, reaction system are controllable, and can generate the signal of stable homogeneous can simultaneously integrate with detection circuit, is closed further
Note.But existing micro-fluidic chip reaction system operates underaction, and reaction signal sensitivity is not high or homogeneity is poor, grasps repeatedly
The convenience of work is poor, cost is difficult to calculate, becomes the main problem of micro-fluidic chip design.
Invention content
The invention provides a kind of micro- based on Electrochemiluminescince fungal detection to solve the problems of the prior art
Fluidic chip, operates flexibly convenient, sensitivity and flexibility is high.
The invention provide based on Electrochemiluminescince fungal detection micro-fluidic chip, including carrying detection unit
Substrate is detected, the detection unit includes connected sample inlet area, memory block, detection zone and the waste of sequence, the inspection
Unit rounded array on the detection substrate is surveyed to arrange and share the sample inlet area, the detection zone in array center
It is loaded with microelectrode so that electrochemical luminescence reaction can be smoothed out.
Further, the detection substrate can be detachably secured in an independent housing, realize detection substrate more
It changes.
Further, the micro-fluidic chip of the invention further includes the detection cover that can be covered on the detection substrate,
The detection cover can be separately provided, it also may be preferable for ground carries out axis connection with the housing by one end.
Wherein, the memory block storage has reagent or labelled antibody, and the detection zone storage has solid phase carrier.
Wherein, the memory block may be configured as interconnected multiple storage regions, between the storage region and deposit
It can further be connected between storage area and detection zone by the shaped form channel of detour.
Wherein, the microelectrode includes working electrode (WE), to electrode (CE) and reference electrode (RE), the work electricity
Pole (WE) can be loaded into electrode (CE) and reference electrode (RE) each independently：A. runner where the detection zone
On bottom surface (for example, when selecting paper as detection substrate material) or the individual paper of b. and it is fixed on the detection zone
In the runner at place or c. is integrated on the corresponding position of the inside of the detection cover, realizes repeatable utilize.
Wherein, the detection cover is equipped with through-hole corresponding with the sample inlet area；With the detection zone position
Corresponding inside is additionally provided with detection window, working electrode, to one kind in electrode, reference electrode, microelectrode pin or more
Kind；The detection lid portion inside is additionally provided with wiring lead, for the transmission of the signals such as light, electricity.
Further, it is described detection cover on be additionally provided with electric signal read and control interface, through the wiring lead respectively with
Each microelectrode or detection window are connected, for detecting the output of signal or testing result.
Further, working electrode described in the microelectrode is by being distributed in the center circle in the detection zone center and around this
Two and half arc A of central both sides are formed, and the center circle is converged with two half arc A to side extension, in its merging end
Portion can further set a working electrode pin；One is provided between the center circle of the working electrode and one of them half arc A
A half arc B is used as to electrode, and half arc C is used as ginseng there are one settings between the center circle of the working electrode and another half arc A
Electrode is examined, it is described that electrode and reference electrode are extended, and in its elongated end to one end opposite with the working electrode pin
Portion further can be respectively set to electrode pin and reference electrode pin.Further, the working electrode pin, to electrode pin
Match with microelectrode pin of the reference electrode pin respectively with being set in the detection cover.
The advantageous effect of the invention is：(1) detection substrate can be replaced, and be repeated for the detection housing of detection
It utilizes, is conducive to cost control；(2) detection unit rounded array arrangement on the detection substrate, it is identical by designing
Detection unit structure can realize that the disposable of same sample repeats to detect, and can also be realized by designing different detection unit structures
Detection project of the same sample under different testing conditions, flexibly convenient, sensitivity and accuracy are high.
Description of the drawings
Fig. 1 is a kind of structure diagram of embodiment of the invention；
Fig. 2 is the structure diagram of Fig. 1 another kind states；
Fig. 3 is the exploded view of Fig. 1；
Fig. 4 is the structure diagram of detection unit in Fig. 1；
Fig. 5 is the structure diagram of microelectrode in Fig. 1；
Fig. 6 is another structure diagram of detection unit in Fig. 1.
In figure, 1- detection substrates；2- detection units；21- sample inlets area；22- memory blocks；221- storage regions；222-
The shaped form channel of detour；23- detection zones；24- wastes；3- microelectrodes；31- working electrodes；311- center circles；Half arcs of 312-
A；32- is to electrode；Half arc B of 321-；33- reference electrodes；Half arc C of 331-；4- housings；5- detects cover；51- through-holes；52- is detected
Window；53- microelectrode pins；54- electric signals are read and control interface.
Specific embodiment
The invention is further described below by with reference to attached drawing.Specific side described in the following examples
Case is not used to the restriction to the invention merely to illustrate the content of the invention.
A kind of preferred embodiment of the invention as shown in Figs. 1-5, includes the detection substrate 1 of carrying detection unit 2,
The detection unit 2 (Fig. 4) includes sequence connected sample inlet area 21, memory block 22, detection zone 23 and waste 24, institute
The rounded array on the detection substrate 1 of detection unit 2 is stated to arrange and share the sample inlet area 21, institute in array center
It states detection zone 23 and is loaded with microelectrode 3 so that electrochemical luminescence reaction can be smoothed out.The detection substrate 1 can be detachable
Ground is fixed in an independent housing 4, realizes the replacement of detection substrate 1.Micro-fluidic chip, which further includes, can be covered in the detection
Detection cover 5 on substrate 1, the detection cover 5 can be separately provided, it also may be preferable for ground is carried out with the housing 4 by one end
Axis connection.
It is described detection substrate 1 be disposable region, selected material can be paper, such as chromatographic paper or
Using plastics etc.；The housing 4 and detection cover 5 are reusable region, and the materials such as plastics, PMMA may be selected, preferably select
High and with certain resistance to corrosion the material with light transmittance.
In the detection unit 2：
Sample inlet area 21 enters the sample containing antigen after processing, and since capillary force acts on, sample will be from sample
Product inlet region 21 is flowed to waste 24,2 size of sample inlet area consider sample-adding convenience and reagent saving and
Paper runner machining accuracy, diameter is 1 to being excellent between 5mm.
Memory block 22 can add in different reagent or labelled antibody thereto according to different testing principles, using sandwich method as
Example, memory block can add in the antibody (powdered) of tris (bipyridine) ruthenium label；When sample flows through the region, generation specific immunity is anti-
It should；Can also memory block 22 may be configured as according to specific situation by interconnected multiple storage regions 221 (Fig. 6), it is described
It can further be connected between storage region 221 and between memory block 22 and detection zone 23 by the shaped form channel 222 of detour,
100~200 μm of channel width, reaches well-mixed purpose；The complexity and dosage of load reagents are considered in memory block 22,
Diameter is preferred in 2mm or so, and it is 50~150 μm that depth can be considered by the dosage of reagent.
The storage of detection zone 23 has solid phase carrier, such as tripropyl amine (TPA) (TPA)；The region needs to load microelectrode 3 simultaneously so that electricity
Chemiluminescence reaction can be smoothed out；The microelectrode 3 includes working electrode 31, to electrode 32 and reference electrode 33, described
Working electrode 31 can be loaded into electrode 32 and reference electrode 33 each independently：A. the runner where the detection zone 23
Bottom surface (for example, when selecting paper as detection substrate material) or the individual paper of b. on and be fixed on the detection
(such as Fig. 1-3) or c. are integrated on the corresponding position of the inside of the detection cover 5 in runner where area 23, and realization can
Recycling.
Working electrode 31 described in the microelectrode 3 (Fig. 5) by the center circle 311 that is distributed in 23 center of the detection zone with
And the two and half arc A 312 around the center both sides are formed, and the center circle 311 and two half arc A extend remittance to side
It closes, a working electrode pin (not shown) can further be set by converging end at it；The center circle of the working electrode 31
There are one half arc B 321 as to electrode 32 for setting between 311 and one of them half arc A 312, in the working electrode 31
The heart sets there are one half arc C 331 to be used as with reference to electrode 33 between justifying 311 and another half arc A 312, described to 32 He of electrode
Reference electrode 33 extends to one end opposite with the working electrode pin, and can further be set respectively in its extended end portion
To electrode pin (not shown) and reference electrode pin (not shown).
In the microelectrode 3, Ag/AgCl can be selected to ensure that reference potential is stablized in reference electrode 33；Working electrode 31 with
And can be selected by graphite electrode (when being integrated on paper), platinum also can be selected since electrode chemical property being needed to stablize for electrode 32
Electrode and gold electrode (when in detection cover 5).
In addition, in the microelectrode 3, working electrode 31 modified (when using magnetic bead as solid phase carrier without into
Row electrode modification), during using Modified ultramicroelectrode, electrode 32 and reference electrode 33 can as previously described will be integrated in simultaneous selection
The inside of the detection cover 5 is to reduce cost.
Waste 24 is mainly used for collecting waste liquid and provides power.During such as using paper as the material for detecting substrate 1, give up
Liquid zone can provide capillary power in itself；During such as using plastics or PMMA as detection 1 material of substrate, it may be selected in detection unit
2 insert chromatographic paper or make micro-column structure, to improve the power that it is provided.
Integral thickness of the detection unit 2 in detection substrate 1 is preferably 100 μm or so, and waste can suitably be deepened.
The detection cover 5 is equipped with and the 21 corresponding through-hole 51 of sample inlet area.
With the corresponding inside in 23 position of detection zone, be additionally provided with detection window 52, detection window 52 can be direct
Into the sampling of traveling optical signal, also can the capture of signal be generated for electrochemical luminescence by electronics integration packaging photoelectric converter.
The selection of photoelectric converter considers the factor of working environment, cost and reliability sensitivity, can select photosensitive electricity
Resistance and photodiode.(wide spectrum responds for photo resistance；Nonpolarity, easy to use, at low cost, long lifespan is simple in structure；
High sensitivity, but far below photomultiplier, operating current is big~mA, easily read)；(high sensitivity is in photosensitive for photodiode
Resistor；High frequency performance is good；Good reliability；It is small, be easily integrated).
Working electrode 31 may also set up in institute one or more of electrode 32, reference electrode 33, microelectrode pin 53
State 5 inside of detection cover；Wherein, the microelectrode pin 53 respectively with the working electrode pin of the microelectrode 3, electrode is drawn
Foot and reference electrode pin match.
Wiring lead (not shown) is additionally provided with inside the detection cover 5, for the transmission of the signals such as light, electricity.
It is described detection cover 5 on be additionally provided with electric signal read with control interface 54, through the wiring lead respectively with it is each micro-
Electrode 3 or detection window 52 are connected, for detecting the output of signal or testing result.
The detection content and flow of several the invention are briefly enumerated below.
1 paper micro-fluidic chip of scheme detects polysaccharide antigen using electrode as solid phase carrier：
1) using microfluidic chip structure as shown in Figs. 1-5, each regional load sample
A) memory block 22 loads the antibody powder of tris (bipyridine) ruthenium label；
B) detection zone 23 loads tripropyl amine (TPA) (TPA)；
C) 23 surface of detection zone prepares the microelectrode of secondary antibody modification by lithographic printing or soft lithography.
2) reaction process：
The sample containing antigen after processing out of chip instills sample inlet area 21 by liquid-transfering gun；Sample flow is stored
During area 22, antigen is combined with labelled antibody, and is enriched on 23 working electrode of detection zone (secondary antibody modification), and subsequent sample will not have
There is the labelled antibody with reference to antigen to pour waste 24.After the enrichment of 5min ,+1V voltages are added on microelectrode 3, are swashed in voltage
It encourages down, in electrode surface redox reaction occurs for chemical substance, emits 620nm photons.
3) testing process：
The photoelectric converter that the photon of reaction transmitting is detected in window 52 is collected, and electric signal is connected everywhere by data line
Circuit (can be realized on PCB or on mobile phone) is managed, filtered device, amplifier filter out noise and amplified signal, utilize mould
Number converter is converted into discrete digital signal by continuous signal is simulated, and the processing modules such as mobile phone, computer is passed to, after data processing
Final result is reflected in interface form on electronic curtain.
Note：Common photoelectric converter has：Photomultiplier, photo resistance and photodiode etc., wherein, photoelectricity times
Increase pipe, need hundreds of to several kilovolts of supply voltages, special equipment is needed to detect, although precision is high, is unfavorable for integrated and reduces
Cost；The spectral response of photo resistance is wide, nonpolarity, at low cost, small, long lifespan, and high sensitivity (is less than photomultiplier transit
Pipe), operating current is big, easily reads；The sensitivity of photodiode is between photomultiplier and photo resistance, high frequency performance
Well, it good reliability, is easily integrated.
2 paper micro-fluidic chip of scheme detects polysaccharide antigen using magnetic bead as solid phase carrier：
1) using the microfluidic chip structure such as scheme 1, detection unit 2 therein is only replaced with to the structure of Fig. 6, each area
Domain sample loading
A) first storage region loads the antibody powder of tris (bipyridine) ruthenium label and the secondary antibody powder of biotin labeling；
B) magnetic bead (magnetized PS pearls) that second storage region loading is modified by Streptavidin；
C) detection zone 23 loads tripropyl amine (TPA) (TPA)；
D) 23 inner surface of detection zone prepares microelectrode 3；
E) 23 lower section integral permanent-magnet iron of detection zone, captures magnetic bead.
2) reaction process：
The sample containing antigen after processing out of chip instills sample inlet area 21 by liquid-transfering gun；It is deposited by first
During storage area, antigen, labelled antibody are combined with label secondary antibody, form interlayer structure；Subsequently enter second memory block, strepto- parent
It is combined with element with biotin, interlayer structure is made to be connected with magnetic bead, and pass through magneticaction and be fixed on electrode.Subsequent samples pair
The structure that detection zone 23 is not associated with antigen plays cleaning action, and flows into waste 24.After the enrichment of 5min, in microelectrode 3
Upper plus+1V voltages, under voltage drive, in electrode surface redox reaction occurs for chemical substance, emits 620nm photons.
3) testing process：
The photoelectric converter that the photon of reaction transmitting is detected in window 52 is collected, and filtered device, amplifier, which filter out, makes an uproar
Sound and amplified signal are converted into discrete digital signal by continuous signal is simulated using analog-digital converter, pass through wired or wireless hair
Receiving device is penetrated, the processing modules such as mobile phone, computer is passed to, final result is reflected in electronics with interface form after data processing
On screen.
3 plastic microfluidic chip of scheme detects polysaccharide antigen using magnetic bead as solid phase carrier：
Basic principle and reagent hold same scheme two, but load the chromatography scraps of paper in waste 24 or prepare microtrabeculae, to
Improve its capillary power, it is ensured that liquid has enough driving forces.
The foregoing is merely the preferred embodiments of the invention, are not intended to limit the invention creation, all at this
Within the spirit and principle of innovation and creation, any modification, equivalent replacement, improvement and so on should be included in the invention
Protection domain within.
Claims (5)
1. one kind is based on Electrochemiluminescince fungal detection micro-fluidic chip, which is characterized in that the inspection including carrying detection unit
Substrate is surveyed, the detection unit includes connected sample inlet area, memory block, detection zone and the waste of sequence, the detection
Unit rounded array on the detection substrate arranges and shares the sample inlet area in array center, and the detection zone adds
It is loaded with microelectrode；
The detection substrate is detachably secured in an independent housing, realizes the replacement of detection substrate；
The micro-fluidic chip further includes the detection cover that can be covered on the detection substrate；The detection cover be equipped with
The corresponding through-hole in the sample inlet area；The corresponding inside of zone position is being detected with described, is being additionally provided with detection window, work electricity
Pole, to one or more in electrode, reference electrode, microelectrode pin；The detection lid portion inside is additionally provided with wiring lead, uses
The transmission of Yu Guang, electric signal.
It is 2. according to claim 1 a kind of based on Electrochemiluminescince fungal detection micro-fluidic chip, which is characterized in that institute
It states multiple storage regions that memory block is set as interconnected, leads between the storage region and between memory block and detection zone
Cross the shaped form channel connection of detour.
It is 3. according to claim 1 a kind of based on Electrochemiluminescince fungal detection micro-fluidic chip, which is characterized in that
The microelectrode includes working electrode, to electrode and reference electrode, the working electrode, to electrode and reference electrode respectively
Independently it is loaded into：A. on the bottom surface of the runner where the detection zone or the individual paper of b. and it is fixed on the inspection
Runner where surveying area is interior or c. is integrated on the corresponding position of the inside for detecting cover.
It is 4. according to claim 1 a kind of based on Electrochemiluminescince fungal detection micro-fluidic chip, which is characterized in that institute
State detection cover on be additionally provided with electric signal read and control interface, the wiring lead respectively with each microelectrode or detection window phase
Even.
It is 5. according to claim 1 a kind of based on Electrochemiluminescince fungal detection micro-fluidic chip, which is characterized in that institute
State working electrode described in microelectrode by be distributed in the center circle in the detection zone center and around the center both sides two and half
Arc A is formed, and the center circle is converged with two half arc A to side extension；The center circle of the working electrode with wherein
There are one half arc B to be used as to electrode for setting between one and half arc A, between the center circle of the working electrode and another half arc A
There are one half arc C to be used as with reference to electrode for setting, it is described to electrode and reference electrode to opposite with the working electrode pin
One end extends.
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| CN105907641B (en) * | 2016-05-19 | 2018-06-29 | 大连医科大学附属第一医院 | A kind of packaging, many condition parallel culture micro fluidic device and its application method |
| CN106018718A (en) * | 2016-07-21 | 2016-10-12 | 武汉市农业科学技术研究院农业环境安全检测研究所 | Multi-component food safety detection microfluidic chip |
| CN107044972A (en) * | 2017-05-25 | 2017-08-15 | 沈阳优宁生物科技有限公司 | A kind of micro-fluidic chip fluorescence immunoassay quick detection kit and its preparation and detection method |
| CN109580506A (en) * | 2018-11-28 | 2019-04-05 | 天津瑞生物科技股份有限公司 | A kind of fungal detection system and two Methods for Fungi Detection based on digital microfluidic technology |
| CN110736776B (en) * | 2019-09-25 | 2020-10-13 | 山东大学 | Urine detection electrochemical sensor, paper diaper and preparation method |
| CN111569959B (en) * | 2020-04-30 | 2021-09-17 | 上海邦先医疗科技有限公司 | Micro-fluidic chip for quantitatively detecting bacteria in biological sample and use method |
| CN119488963A (en) * | 2024-10-21 | 2025-02-21 | 桂林电子科技大学 | A microfluidic chip that integrates enrichment function and electrochemiluminescence detection |
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